Polypeptides having glucoamylase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to polypeptides having glucoamylase activity and isolated polynucleotides encoding said polypeptides preferably derived from a strain of  Peniphora rufomarginata . The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides. The invention also relates to the composition comprising a glucoamylase of the invention as well as the use such compositions for starch conversion processes, brewing, including processes for producing fermentation products or syrups.

CROSS-REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to polypeptides having glucoamylaseactivity and polynucleotides encoding the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the polynucleotides as well as methods for producing andusing the polypeptides, and to the use of glucoamylases of the inventionfor starch conversion to producing fermentation products, such asethanol, and syrups, such as glucose. The invention also relates to acomposition comprising a glucoamylase of the invention.

BACKGROUND OF THE INVENTION

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is anenzyme, which catalyzes the release of D-glucose from the non-reducingends of starch or related oligo- and polysaccharide molecules.Glucoamylases are produced by several filamentous fungi and yeast, withthose from Aspergillus being commercially most important.

Commercially, glucoamylases are used to convert starchy material, whichis already partially hydrolyzed by an alpha-amylase, to glucose. Theglucose may then be converted directly or indirectly into a fermentationproduct using a fermenting organism. Examples of commercial fermentationproducts include alcohols (e.g., ethanol, methanol, butanol,1,3-propanediol); organic acids (e.g., citric acid, acetic acid,itaconic acid, lactic acid, gluconic add, gluconate, lactic add,succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone);amino acids (e.g., glutamic acid), gases (e.g., H₂ and CO₂), and morecomplex compounds, including, for example, antibiotics (e.g., penicillinand tetracycline); enzymes: vitamins (e.g., riboflavin, B₁₂,beta-carotene), hormones, and other compounds which are difficult toproduce synthetically. Fermentation processes are also commonly used inthe consumable alcohol (e.g., beer and wine), dairy (e.g., in theproduction of yoghurt and cheese), leather, and tobacco industries.

The end product may also be syrup. For instance, the end product may beglucose, but may also be converted, e.g., by glucose isomerase tofructose or a mixture composed almost equally of glucose and fructose.This mixture, or a mixture further enriched with fructose, is the mostcommonly used high fructose corn syrup (HFCS) commercialized throughoutthe world.

Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102 disclose Aspergillusniger G1 or G2 glucoamylase.

U.S. Pat. No. 4,727,046 discloses a glucoamylase derived from Corticiumrolfsii which is also referred to as Athelia rolfsii.

WO 84/02921 discloses a glucoamylase derived from Aspergillus awamori.

WO 99/28248 discloses a glucoamylase derived from Talaromyces emersonii.

WO 00/75296 discloses a glucoamylase derived from Thermoascuscrustaceus.

WO 2006/069289 discloses glucoamylases derived from Trametes cingulate,Pachykytospora papyracea, and Leucopaxillus giganteus.

It is an object of the present invention to provide polypeptides havingglucoamylase activity and polynucleotides encoding the polypeptides andwhich provide a high yield in fermentation product production processes,such as ethanol production processes, including one-step ethanolfermentation processes from un-gelatinized raw (or uncooked) starch.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides having glucoamylaseactivity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 60%identity with amino acids for mature polypeptide amino acids 1 to 558 ofSEQ ID NO: 2;

(b) a polypeptide which is encoded by a nucleotide sequence (i) whichhybridizes under at least low stringency conditions with nucleotides 61to 2301 of SEQ ID NO: 1, or (ii) which hybridizes under at least lowstringency conditions with the cDNA sequence contained in nucleotides 61to 1734 of SEQ ID NO: 3, or (iii) a complementary strand of (i) or (ii);

(c) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of amino acids 1 to 558 of SEQ IDNO: 2.

The present invention also relates to polynucleotides encodingpolypeptides having glucoamylase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 60% identity with the mature polypeptideamino acids 1 to 558 of SEQ ID NO: 2;

(b) a polynucleotide having at least 60% identity with nucleotides 61 to2301 of SEQ ID NO: 1; or

(c) a polynucleotide having at least 60% identity with nucleotides 61 to1734 of SEQ ID NO: 3;

(d) a polypeptide which is encoded by a nucleotide sequence (i) whichhybridizes under at least low stringency conditions with nucleotides 61to 2301 of SEQ ID NO: 1, or (ii) which hybridizes under at least lowstringency conditions with the cDNA sequence contained in nucleotides 61to 1734 of SEQ ID NO: 3, or (iii) a complementary strand of (i) or (ii).

In a preferred embodiment the polypeptide is derivable from a strain ofthe genus Peniphora, preferably a strain of the species Peniphorarufomarginata or E. coli strain deposited at DSMZ on 3 Apr. 2006 underthe terms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure atDeutshe Sammmlung von Microorganismen and Zeilkulturen GmbH (DSMZ),Mascheroder Weg 1b, D-38124 Braunschweig DE. The clone was given the no.DSM 18150. Deposited strain DSM 18150 harbors plasmid pENI2516comprising a sequence that, to the best belief of the inventors, isidentical to SEQ ID NO: 1. A specific polypeptide of the invention isthe mature polypeptide obtained when expressing plasmid pENI2516 in asuitable fungal host cell.

The present invention also relates to methods for producing suchpolypeptides having glucoamylase activity comprising (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide, and (b) recovering the polypeptide.

The present invention also relates to processes of producingfermentation products or syrups.

DEFINITIONS

Glucoamylase activity: The term glucoamylase (1,4-alpha-D-glucanglucohydrolase, EC 3.2.1.3) is defined as an enzyme, which catalyzes therelease of D-glucose from the non-reducing ends of starch or relatedoligo- and polysaccharide molecules. For purposes of the presentinvention, glucoamylase activity is determined according to theprocedure described in the “Materials & Methods”-section below.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the glucoamylase activity of thepolypeptide consisting of the amino acid sequence shown as amino acids 1to 558 of SEQ ID NO: 2.

Polypeptide: The term “polypeptide” as used herein refers to an isolatedpolypeptide which is at least 20% pure, preferably at least 40% pure,more preferably at least 60% pure, even more preferably at least 80%pure, most preferably at least 90% pure, and even most preferably atleast 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, at most 3%, even morepreferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polypeptide material withwhich it is natively associated. It is, therefore, preferred that thesubstantially pure polypeptide is at least 92% pure, preferably at least94% pure, more preferably at least 95% pure, more preferably at least96% pure, more preferably at least 96% pure, more preferably at least97% pure, more preferably at least 98% pure, even more preferably atleast 99%, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form”.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR. Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASEGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of SEQ ID NO: 2, or homologous sequencesthereof, wherein the fragment has glucoamylase activity.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5 and/or 3′ endof SEQ ID NO: 1 or 3, or homologous sequences thereof, wherein thesubsequence encodes a polypeptide fragment having glucoamylase activity.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e. that the polynucleotide preparationis essentially free of other polynucleotide material with which it isnatively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semi-synthetic, synthetic origin, or anycombinations thereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,pro-peptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may a DNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct comprising a polynucleotide ofthe present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the amino acids 1 to 558of SEQ ID NO: 2, as well as genetic manipulation of the DNA encoding thepolypeptides. The modification(s) can be substitution(s), deletion(s)and/or insertions(s) of the amino acid(s) as well as replacement(s) ofamino acid side chain(s).

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having glucoamylase activity produced by an organismexpressing a modified nucleotide sequence of SEQ ID NOS: 1 (genomic DNA)or 3 (cDNA). The modified nucleotide sequence is obtained through humanintervention by modification of the nucleotide sequence disclosed in SEQID NO: 1 or 3.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having GlucoamylaseActivity

In a first aspect, the present invention relates to polypeptides havingan amino acid sequence which has a degree of identity to amino acids 1to 558 of SEQ ID NO: 2 (i.e. mature polypeptide).

In an embodiment the polypeptide is a variant comprising a conservativesubstitution, deletion, and/or insertion of one or more amino acids ofamino acids 1 to 558 of SEQ ID NO: 2.

In an embodiment the amino acid sequence has glucoamylase activity andis at least 60%, preferably at least 70%, preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, more preferred at least 96%, even morepreferred at least 97%, even more preferred at least 98%, even morepreferably at least 99% identical to the mature part of SEQ ID NO: 2(hereinafter “homologous polypeptides”).

In a preferred aspect, the homologous polypeptides have an amino acidsequence which differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from amino acids 1 to 558 of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises the matureamino acid sequences of SEQ ID NO: 2, or allelic variants thereof; orfragments thereof that have glucoamylase activity, e.g., the catalyticdomain.

Catalytic Domain

In an aspect, the invention relates to polypeptides that comprise thecatalytic region/domain of the amino acid sequences of SEQ ID NO: 2.

The catalytic region/domain of the invention exhibiting glucoamylaseactivity, preferably derived from a strain of Peniophora, especially astrain of preferably Peniophora ruromarginata, is located from aminoacids 1 to 448 in SEQ ID NO: 2. In one embodiment the region may beconsidered to include the linker region from amino acids 449 to 463 ofSEQ ID NO: 2, or part thereof. The putative binding domain is encoded bypolynucleotides 1845 to 2301 in SEQ ID NO: 1 or polynucleotides1450-1734 of SEQ ID NO: 3.

In a preferred embodiment the invention relates to a catalytic regionwhich has at least 60% identity, preferably at least 65% identity, morepreferably at least 70% identity, more preferably at least 75% identity,more preferably at least 80% identity, more preferably at least 85%identity, even more preferably at least 90% identity, most preferably atleast 95% identity, more preferred at least 96% identity, even morepreferred at least 97% identity, even more preferred at least 98%identity, even more preferably at least 99% identity, especially 100%identity to amino acids 1 to 448 in SEQ ID NO: 2, and which haveglucoamylase activity (hereinafter “homologous polypeptides”). In apreferred aspect, the homologous catalytic regions have amino acidsequences which differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from amino acids 1 to 448 of SEQ ID NO: 2.

Binding Domain

In another aspect, the invention relates to polypeptides havingcarbohydrate-binding affinity, preferably starch-binding affinity.

The binding domain in Peniophora ruformarginata glucoamylase is locatedfrom amino acid 464 to 558 of SEQ ID NO: 2 and is encoded bypolynucleotides 1845-2301 in SEQ ID NO: 1 or 1450-1734 of SEQ ID NO: 3.

Consequently, in this aspect the invention relates to a polypeptidehaving carbohydrate-binding affinity, selected from the group consistingof:

(a) i) a polypeptide comprising an amino acid sequence which has atleast 60% identity with amino acids 464 to 558 of SEQ ID NO: 2;(b) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under low stringency conditions with a polynucleotide probewhich has the complementary strand of nucleotides 1845 to 2301 of SEQ IDNO: 1 or nucleotides 1450 to 1734 of SEQ ID NO: 3, respectively;(c) a fragment of (a) or (b) that has carbohydrate binding affinity.

In a preferred embodiment the carbohydrate binding affinity isstarch-binding affinity.

In a preferred embodiment the invention relates to a polypeptide havingcarbohydrate binding affinity which has at least 60% identity,preferably at least 70% identity, more preferably at least 75% identity,more preferably at least 80% identity, more preferably at least 85%identity, even more preferably at least 90% identity, most preferably atleast 95% identity, more preferred at least 96% identity, even morepreferred at least 97% identity, even more preferred at least 98%identity, even more preferably at least 99% identity, especially 100%identity to amino acids 464 to 558 in SEQ ID NO: 2.

In a preferred aspect, homologous binding domains have amino acidsequences which differ by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from amino acids 464 to 558 of SEQ ID NO:2.

The invention also relates to a polypeptide having carbohydrate-bindingaffinity, where the polypeptide is an artificial variant which comprisesan amino acid sequence that has at least one substitution, deletionand/or insertion of an amino acid as compared to amino acids 464 to 558of SEQ ID NO: 2.

The invention also relates to a polypeptide having carbohydrate-bindingaffinity, where the polypeptide is an artificial variant which comprisesan amino acid sequence that has at least one substitution, deletionand/or insertion of an amino acid as compared to the amino acid sequenceencoded by the carbohydrate-binding domain encoding part of thepolynucleotide sequences shown in position 1845-2301 in SEQ ID NO: 1, or1450 to 1734 in SEQ ID NO: 3.

Hybrids

The glucoamylases or catalytic regions of the invention may be linked,via a linker sequence or directly, to one or more foreign bindingdomains (also referred to as binding modules (CBM)). A “foreign” bindingdomain is a binding-domain that is not derived from the wild-typeglucoamylase of the invention. The binding-domain is preferably acarbohydrate-binding domain (i.e., having affinity for binding to acarbohydrate), especially a starch-binding domain or a cellulose-bindingdomain. Preferred binding domains are of fungal or bacterial origin.Examples of specifically contemplated starch-binding domains aredisclosed in WO 2005/003311 which is hereby incorporated by reference.

In a preferred embodiment the linker in a glucoamylase of the inventionis replaced with a more stable linker, i.e., a linker that is moredifficult to cut than the parent linker. This is done to avoid that thebinding-domain is cleaved off. Specifically contemplated stable linkersinclude the Aspergillus kawachii linker:

TTTTTTAAAT STSKATTSSSSSSAAATTSSS (SEQ ID NO: 4)

Thus, in a preferred embodiment the invention relates to a hybridglucoamylase having the amino acid sequence shown in SEQ ID NO: 2,wherein the native linker located from amino acids 449 to 463 of SEQ IDNO: 2, or part thereof, is replaced with the Aspergillus kawachii linkershown in SEQ ID NO: 4.

Thus, the invention also relates to hybrids consisting of a glucoamylaseof the invention or catalytic domain of the invention havingglucoamylase activity fused to a stable linker (e.g., Aspergilluskawachii linker) and one or more carbohydrate-binding domains, e.g., acarbohydrate-binding module (CBM) disclosed in WO 2005/003311 on page 5,line 30 to page 8, line 12, hereby incorporated by reference.

Hybridization

In another aspect, the present invention relates to polypeptides havingglucoamylase activity which are encoded by polynucleotides (i) whichhybridizes under at least low stringency conditions, preferably mediumstringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with a nucleotide sequencewith nucleotides 61 to 2301 of SEQ ID NO: 1 (Peniophora genomic DNA) ornucleotides 61 to 1734 of SEQ ID NO: 3 (Peniophora cDNA), or (ii) asubsequence of (i), or (iii) a complementary strand of (i) or (ii) (J.Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequenceof SEQ ID NOS: 1 or 3 contains at least 100 contiguous nucleotides orpreferably at least 200 continguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has glucoamylaseactivity.

The nucleotide sequence of SEQ ID NOS: 1 or 3, or a subsequence thereof,as well as the amino acid sequence of SEQ ID NO: 2, or a fragmentthereof, may be used to design a nucleic acid probe to identify andclone DNA encoding polypeptides having glucoamylase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes which are at least 600 nucleotides, atleast preferably at least 700 nucleotides, more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³P, ³H, ³⁵S, biotin,or avidin). Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having glucoamylaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NOS: 1 or 3, or a subsequence thereof, thecarrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequences hybridize to labeled nucleic acid probescorresponding to the nucleotide sequence shown in SEQ ID NOS: 1 or 3,its complementary strands, or subsequences thereof, under low to veryhigh stringency conditions, Molecules to which the nucleic acid probehybridizes under these conditions can be detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is nucleotides 61 to2301 of SEQ ID NO: 1 or nucleotides 61 to 1734 of SEQ ID NO: 3. Inanother preferred aspect, the nucleic acid probe is a polynucleotidesequence which encodes the catalytic region between amino acids 1-448 ofSEQ ID NO: 2.

In another aspect the invention relates to nucleic acid probes thatencode the binding domain in amino acids 464 to 558 of SEQ ID NO: 2.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding region of SEQ ID NOS: 1 or 3, respectively.

In another preferred aspect, the nucleic acid probe is the part of thesequences in plasmid pENI2516 coding for the mature polypeptides of theinvention. Plasmid pENI2516 which are contained in Escherichia coli DSM18150 encode polypeptides having glucoamylase activity.

For long probes of at least 100 nucleotides in length, low to very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micro g/ml sheared and denaturedsalmon sperm DNA, and either 25% formamide for low stringencies, 35%formamide for medium and medium-high stringencies, or 50% formamide forhigh and very high stringencies, following standard Southern blottingprocedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 50° C. (low stringency), more preferablyat least at 55° C. (medium stringency), more preferably at least at 60°C. (medium-high stringency), even more preferably at least at 65° C.(high stringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

Under salt-containing hybridization conditions, the effective T_(m) iswhat controls the degree of identity required between the probe and thefilter bound DNA for successful hybridization. The effective T_(m) maybe determined using the formula below to determine the degree ofidentity required for two DNAs to hybridize under various stringencyconditions.

Effective T _(m)=81.5+16.6(log M[Na⁺])+0.41(% G+C)−0.72(% formamide)

(See www.ndsu.nodak.edu/insruct/mcclean/plsc731/dna/dna6.htm)

Variants

In a further aspect, the present invention relates to artificialvariants comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids in SEQ ID NO: 2, or the maturepolypeptide thereof. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions or insertions thatdo not significantly affect the folding and/or activity of the protein;small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline,

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,glucoamylase activity) to identify amino acid residues that are criticalto the activity of the molecule. See also, Hilton et al. 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzymes or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312, Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64.The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides which are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57: Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 2152-2156; WO 95/17413; or WO 95/22625.Other methods that can be used include error-prone PCR, phage display(e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat. No.5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire etal. 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The total number of amino acid substitutions, deletions and/orinsertions of amino acids in position 1 to 558 of SEQ ID NO: 2, is 10,preferably 9, more preferably 8, more preferably 7, more preferably atmost 6, more preferably at most 5, more preferably 4, even morepreferably 3, most preferably 2, and even most preferably 1.

Sources of Polypeptides Having Glucoamylase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

In a preferred embodiment, the glucoamylase of the invention derivedfrom the class Basidiomycetes. In a more preferred embodiment aglucoamylase of the invention is derived from a strain of the genusPeniophora, more preferably from a strain of the species Peniophorareformarginata, or deposited as Escherichia coli clone DSM 18150.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

The Peniophora refomarginata strain was collected in Denmark in 1997.

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of another microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.In a preferred aspect, the nucleotide sequence is set forth in any ofSEQ ID NO: 1 (genomic DNA) or 3 (cDNA), respectively. In another morepreferred aspect, the nucleotide sequence is the sequence contained inplasmid pENI2516 that is contained in Escherichia coli DSM 18150 Inanother preferred aspect, the nucleotide sequence is the maturepolypeptide coding region of any of SEQ ID NOS: 1 or 3, respectively.The present invention also encompasses nucleotide sequences which encodea polypeptide having the amino acid sequence of SEQ ID NO: 2, or themature polypeptide thereof, which differs from SEQ ID NOS: 1 or 3,respectively, by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of any of SEQ ID NOS: 1or 3, respectively, which encode fragments of SEQ ID NO: 2 that haveglucoamylase activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of anyof SEQ ID NOS: 1 or 3, respectively, in which the mutant nucleotidesequence encodes a polypeptide which consists of amino acids 1 to 558 ofSEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from any organism, especially a strain of the genusPeniophora or other or related organisms and thus, for example, may bean allelic or species variant of the polypeptide encoding region of thenucleotide sequences.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 (i.e. nucleotides 61 to 2301), or SEQ IDNO: 3 (i.e., nucleotides 61 to 1734), respectively, of at least 60%,preferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, more preferably at least 90%, even more preferably at least 95%,even more prefer ably 96%, even more 97%, even more 98%, and mostpreferably at least 99% identity, which encode an active polypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide encoding regionof any of SEQ ID NOS: 1 or 3, respectively, e.g. subsequences thereof,and/or by introduction of nucleotide substitutions, which do not giverise to another amino acid sequence of the polypeptide encoded by thenucleotide sequence, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions which may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forglucoamylase activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-enzyme interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al. 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, (i) which hybridize under lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with nucleotides 61 to 2301 of SEQ ID NO: 1 or nucleotides 61to 1734 of SEQ ID NO: 3, respectively, or (ii) a complementary strand of(i); or allelic variants and subsequences thereof (Sambrook et al. 1989,supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under low, medium, medium-high,high, or very high stringency conditions with (i) nucleotides 61 to 2301of SEQ ID NO: 1 or nucleotides 61 to 1734 of SEQ ID NO: 3, respectively,or (ii) a complementary strand of (i); and (b) Isolating the hybridizingpolynucleotide, which encodes a polypeptide having glucoamylaseactivity,

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (gfaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase. Aspergillus nidulans acetamidase, Fusariumvenenatum glucoamylase (WO 00/56900), Fusarium venenatum Daria (WO00/56900). Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidaseTrichoderma reesei cellobiohydrolase I, Trichoderma reesei endoglucanaseI, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1). Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase.Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase, Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the TAKA alpha-amylase promoter, Aspergillus nigerglucoamylase promoter, and Aspergillus oryzae glucoamylase promoter maybe used as regulatory sequences. Other examples of regulatory sequencesare those which allow for gene amplification. In eukaryotic systems,these include the dihydrofolate reductase gene which is amplified in thepresence of methotrexate, and the metallothionein genes which areamplified with heavy metals. In these cases, the nucleotide sequenceencoding the polypeptide would be operably linked with the regulatorysequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

Examples of suitable markers for yeast host cells are ADE2, HIS3, LEU2,LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentousfungal host cell include, but are not limited to, amdS (acetamidase),argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell are theamdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae andthe bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., in, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes) Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A. Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteria Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces Saccharomyces kluyveri, Saccharomyces norbensis orSaccharomyces oviformis cell. In another most preferred aspect, theyeast host cell is a Kluyveromyces lactis cell. In another mostpreferred aspect, the yeast host cell is a Yarrowia lipotytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth at, 1995,supra). The filamentous fungi are generally characterized by a mycelialwall composed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,Aspergillus kawachii, or Aspergillus oryzae cell. In another mostpreferred aspect, the filamentous fungal host cell is a Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioictes, or Fusarium venenatum cell. In another mostpreferred aspect, the filamentous fungal host cell is a Bjerkanderaadusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsiscaregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta,Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Ceriporiopsissubvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola isolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crease, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiate, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma ressei,or Trichoderma viride strain cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York: Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a cell,which in its wild-type form is capable of producing the polypeptide,under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. Preferably, the cell is a strain of thegenus Peniophora, more preferably a strain of the species Peniophorarufomarginata.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a nucleotide sequence having the maturepolypeptide coding region of SEQ ID NOS: 1 or 3, respectively, whereinthe nucleotide sequence encodes a polypeptide which consists of aminoacids 1 to 558 of SEQ ID NO: 2, and (b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having glucoamylase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal, 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev, Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Viola faba promoterfrom the legumin B4 and the unknown seed protein gene from Viola faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mita and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g. ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having glucoamylase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theglucoamylase activity of the composition has been increased, e.g., by anenrichment factor of 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae, Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Combination of Glucoamylase and Acid Alpha-Amylase

According to this aspect of the invention a glucoamylase of theinvention may be combined with an alpha-amylase, preferably acidalpha-amylase in a ratio of between 0.3 and 5.0 AFAU/AGU. Morepreferably the ratio between acid alpha-amylase activity andglucoamylase activity is at least 0.35, at least 0.40, at least 050, atleast 0.60, at least 0.7, at least 0.8, at least 0.9, at least 1.0, atleast 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, atleast 1.6, at least 1.7, at least 1.8, at least 1.85, or even at least1.9 AFAU/AGU. However, the ratio between acid alpha-amylase activity andglucoamylase activity should preferably be less than 4.5, less than 4.0,less than 3.5, less than 3.0, less than 25, or even less than 2.25AFAU/AGU. In AUU/AGI the activities of acid alpha-amylase andglucoamylase are preferably present in a ratio of between 0.4 and 6.5AUU/AGI. More preferably the ratio between acid alpha-amylase activityand glucoamylase activity is at least 0.45, at least 0.50, at least0.60, at least 0.7, at least 08, at least 0.9, at least 1.0, at least1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least2.1, at least 2.2, at least 2.3, at least 2.4, or even at least 2.5AUU/AGI, However, the ratio between acid alpha-amylase activity andglucoamylase activity is preferably less than 6.0, less than 55, lessthan 4.5, less than 4.0, less than 3.5, or even less than 3.0 AUU/AGI.

Above composition is suitable for use in a starch conversion processmentioned below for producing syrup and fermentation products, such asethanol.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to processes/methods for usingthe polypeptides having glucoamylase activity of the invention.

Uses according to the invention include starch conversion of starch toe.g., syrup and fermentation products, including ethanol and beverages.Examples of processes where a glucoamylase of the invention may be usedinclude the ones described in: WO 2004/081193, WO 2004/080923, WO2003/66816, WO 2003/66826, and WO 92/20777 which are hereby allincorporated by reference.

Production of Fermentation Products

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the present invention relates to a process for producinga fermentation product, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.

The invention relates to a process for producing a fermentation productfrom starch-containing material comprising the steps of:

(a) liquefying starch-containing material;

(b) saccharifying the liquefied material obtained in step (a) using aglucoamylase of the invention;

(c) fermenting the saccharified material using a fermenting organism.

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. Suitablestarch-containing starting materials are listed in the section“Starch-containing materials”-section below. Contemplated enzymes arelisted in the “Enzymes”-section below. The liquefaction is preferablycarried out in the presence of an alpha-amylase. The fermentation ispreferably carried out in the presence of yeast, preferably a strain ofSaccharomyces. Suitable fermenting organisms are listed in the“Fermenting Organisms”-section below. In preferred embodiments step (b)and (c) are carried out sequentially or simultaneously (i.e., as SSFprocess).

In a particular embodiment, the process of the invention furthercomprises, prior to the step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling:

y) forming a slurry comprising the starch-containing material and water.

The aqueous slurry may contain from 10-40 wt-%, preferably 25-35 wt-%starch-containing material. The slurry is heated to above thegelatinization temperature and alpha-amylase, preferably bacterialand/or acid fungal alpha-amylase, may be added to initiate liquefaction(thinning). The slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to an alpha-amylase in step(a) of the invention.

More specifically liquefaction may be carried out as a three-step hotslurry process. The slurry is heated to between 60-95° C., preferably80-85° C., and alpha-amylase is added to initiate liquefaction(thinning). Then the slurry may be jet-cooked at a temperature between95-140° C., preferably 105-125° C. for 1-15 minutes, preferably for 3-10minute, especially around 5 minutes. The slurry is cooled to 60-95° C.and more alpha-amylase is added to finalize hydrolysis (secondaryliquefaction). The liquefaction process is usually carried out at pH4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefiedwhole grains are known as mash.

The saccharification in step (b) may be carried out using conditionswell know in the art. For instance, a full saccharification process maylast up to from about 24 to about 72 hours, however, it is common onlyto do a pre-saccharification of typically 40-90 minutes at a temperaturebetween 30-65° C., typically about 60° C., followed by completesaccharification during fermentation in a simultaneous saccharificationand fermentation process (SSF process). Saccharification is typicallycarried out at temperatures from 30-65° C., typically around 60° C., andat a pH between 4 and 5, normally at about pH 4.5.

The most widely used process in fermentation product, especiallyethanol, production is the simultaneous saccharification andfermentation (SSF) process, in which there is no holding stage for thesaccharification, meaning that fermenting organism, such as yeast, andenzyme(s) may be added together. SSF may typically be carried out at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In accordance with the present invention the fermentation step (c)includes, without limitation, fermentation processes used to producealcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citricacid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones(e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g. H₂ andCO₂); antibiotics (e.g., penicillin and tetracycline): enzymes; vitamins(e.g., riboflavin, B12, beta-carotene); and hormones. Preferredfermentation processes include alcohol fermentation processes, as arewell known in the art. Preferred fermentation processes are anaerobicfermentation processes, as are well known in the art.

Processes for Producing Fermentation Products from Un-GelatinizedStarch-Containing

In this aspect the invention relates to processes for producing afermentation product from starch-containing material withoutgelatinization of the starch-containing material. In one embodiment onlya glucoamylase of the invention is used during saccharification andfermentation. According to the invention the desired fermentationproduct, such as ethanol, can be produced without liquefying the aqueousslurry containing the starch-containing material. In one embodiment aprocess of the invention includes saccharifying (milled)starch-containing material e.g., granular starch, below thegelatinization temperature in the presence of a glucoamylase of theinvention to produce sugars that can be fermented into the desiredfermentation product by a suitable fermenting organism.

Example 4 below discloses production of ethanol from un-gelatinized(uncooked) milled corn using glucoamylases of the invention derived fromPeniphora rufomarginata for one-step fermentation alone and incombination with an alpha-amylase.

Accordingly, in this aspect the invention relates to a process forproducing a fermentation product from starch-containing materialcomprising:

(a) saccharifying starch-containing material with a glucoamylase having

the sequence shown as amino acids 1 to 558 in SEQ ID NO: 2, or aglucoamylase having at least 60% identity thereto,

at a temperature below the initial gelatinization temperature of saidstarch-containing material,

(b) fermenting using a fermenting organism.

Steps (a) and (b) of the process of the invention may be carried outsequentially or simultaneously. In an embodiment a slurry comprisingwater and starch-containing material is prepared before step (a).

The fermentation process may be carried out for a period of 1 to 250hours, preferably is from 25 to 190 hours, more preferably from 30 to180 hours, more preferably from 40 to 170 hours, even more preferablyfrom 50 to 160 hours, yet more preferably from 60 to 150 hours, even yetmore preferably from 70 to 140 hours, and most preferably from 80 to 130hours.

The term “initial gelatinization temperature” means the lowesttemperature at which gelatinization of the starch commences. Starchheated in water begins to gelatinize between 50° C. and 75° C.; theexact temperature of gelatinization depends on the specific starch, andcan readily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material is the temperature atwhich birefringence is lost in 5% of the starch granules using themethod described by Gorinstein. S. and Lii. C., Starch/Stärke, Vol. 44(12) pp, 461-466 (1992).

Before step (a) a slurry of starch-containing material, such as granularstarch, having 10-55 wt.-% dry solids, preferably 25-40 wt.-% drysolids, more preferably 30-35% dry solids of starch-containing materialmay be prepared. The slurry may include water and/or process waters,such as stillage (backset), scrubber water, evaporator condensate ordistillate, side stripper water from distillation, or other fermentationproduct plant process water. Because the process of the invention iscarried out below the gelatinization temperature and thus no significantviscosity increase takes place, high levels of stillage may be used ifdesired. In an embodiment the aqueous slurry contains from about 1 toabout 70 vol.-% stillage, preferably 15-60% vol.-% stillage, especiallyfrom about 30 to 50 vol.-% stillage.

The starch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids of the starch-containing material is convertedinto a soluble starch hydrolysate.

The process of the invention is conducted at a temperature below theinitial gelatinization temperature. Preferably the temperature at whichstep (a) is carried out is between 30-75° C., preferably between 45-60°C.

In a preferred embodiment step (a) and step (b) are carried out as asequential or simultaneous saccharification and fermentation process. Insuch preferred embodiment the process is typically carried at atemperature between 25° C. and 40° C., such as between 29° C. and 35°C., such as between 30° C. and 34° C., such as around 32° C. Accordingto the invention the temperature may be adjusted up or down duringfermentation.

In an embodiment simultaneous saccharification and fermentation iscarried out so that the sugar level, such as glucose level, is kept at alow Level such as below 6 wt.-%, preferably below about 3 wt.-%,preferably below about 2 wt.-%, more preferred below about 1 wt.-%.,even more preferred below about 0.5%, or even more preferred 0.25%wt.-%, such as below about 0.1 wt.-%. Such low levels of sugar can beaccomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich quantities of enzyme and fermenting organism to use. The employedquantities of enzyme and fermenting organism may also be selected tomaintain low concentrations of maltose in the fermentation broth. Forinstance, the maltose level may be kept below about 0.5 wt.-% or belowabout 0.2 wt.-%.

The process of the invention may be carried out at a pH in the rangebetween 3 and 7, preferably from pH 3.5 to 6, or more preferably from pH4 to 5.

Starch-Containing Materials

Any suitable starch-containing starting material, including granularstarch, may be used according to the present invention. The startingmaterial is generally selected based on the desired fermentationproduct. Examples of starch-containing starting maters, suitable for usein a process of present invention, include tubers, roots, sterns, wholegrains, corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca,sorghum, rice peas, beans, or sweet potatoes, or mixtures thereof, orcereals, sugar-containing raw materials, such as molasses, fruitmaterials, sugar cane or sugar beet, potatoes, and cellulose-containingmaterials, such as wood or plant residues, or mixtures thereof.Contemplated are both waxy and non-waxy types of corn and barley.

The term “granular starch” means raw uncooked starch, i.e., starch inits natural form found in cereal, tubers or grains. Starch is formedwithin plant cells as tiny granules insoluble in water. When put in coldwater, the starch granules may absorb a small amount of the liquid andswell. At temperatures up to 50° C. to 75° C. the swelling may bereversible. However, with higher temperatures an irreversible swellingcalled “gelatinization” begins. Granular starch to be processed may be ahighly refined starch quality, preferably at least 90%, at least 95%, atleast 97% or at least 99.5% pure or it may be a more crude starchcontaining material comprising milled whole grain including non-starchfractions such as germ residues and fibers. The raw material, such aswhole grain, is milled in order to open up the structure and allowingfor further processing. Two milling processes are preferred according tothe invention: wet and dry milling. In dry milling whole kernels aremilled and used. Wet milling gives a good separation of germ and meal(starch granules and protein) and is often applied at locations wherethe starch hydrolysate is used in production of syrups. Both dry and wetmilling is well known in the art of starch processing and is equallycontemplated for the process of the invention.

The starch-containing material is reduced in particle size, preferablyby dry or wet milling, in order to expose more surface area. In anembodiment the particle size is between 0.05 to 3.0 mm, preferably0.1-0.5 mm, or so that at least 30%, preferably at least 50%, morepreferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.ethanol, methanol, butanol); organic acids (e.g., citric acid, aceticacid, itaconic acid, lactic acid, gluconic acid); ketones (e.g.,acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂);antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin. B₁₂, beta-carotene); and hormones. In a preferredembodiment the fermentation product is ethanol, e.g., fuel ethanol;drinking ethanol, i.e., potable neutral spirits; or industrial ethanolor products used in the consumable alcohol industry (e.g., beer andwine), dairy industry (e.g., fermented dairy products), leather industryand tobacco industry. Preferred beer types comprise ales, stouts,porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer,low-alcohol beer, low-calorie beer or light beer. Preferred fermentationprocesses used include alcohol fermentation processes, as are well knownin the art. Preferred fermentation processes are anaerobic fermentationprocesses, as are well known in the art.

Fermenting Organisms

“Fermenting organism” refers to any organism, including bacterial andfungal organisms, suitable for use in a fermentation process and capableof producing desired a fermentation product. Especially suitablefermenting organisms are able to ferment, i.e., convert, sugars, such asglucose or maltose, directly or indirectly into the desired fermentationproduct. Examples of fermenting organisms include fungal organisms, suchas yeast. Preferred yeast includes strains of Saccharomyces spp., inparticular, Saccharomyces cerevisiae. Commercially available yeastinclude, e.g., Red Star™/Lesaffre Ethanol Red (available from RedStar/Lesaffre, USA) FALI (available from Fleischmann's Yeast, a divisionof Burns Philp Food inc., USA), SUPERSTART (available from Alltech),GERT STRAND (available from Gert Strand AB, Sweden) and FERMIOL(available from DSM Specialties).

Enzymes Glucoamylase

The glucoamylase is preferably a glucoamylase of the invention. However,as mentioned above a glucoamylase of the invention may also be combinedwith other glucoamylases.

The glucoamylase may added in an amount of 0.001 to 10 AGU/g DS,preferably from 0.01 to 5 AGU/g DS, such as around 0.1, 0.3, 0.5, 1 or 2AGU/g DS, especially 0.1 to 0.5 AGU/g DS or 0.02-20 AGU/g DS, preferably0.1-10 AGU/g DS.

Alpha-Amylase

The alpha-amylase may according to the invention be of any origin.Preferred are alpha-amylases of fungal or bacterial origin.

In a preferred embodiment the alpha-amylase is an acid alpha-amylase,e.g., fungal acid alpha-amylase or bacterial acid alpha-amylase. Theterm “acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) whichadded in an effective amount has activity optimum at a pH in the rangeof 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylases

According to the invention a bacterial alpha-amylase may preferably bederived from the genus Bacillus.

In a preferred embodiment the Bacillus alpha-amylase is derived from astrain of B. licheniformis, B. amyloliquefaciens, B. subtilis or B.stearothermophilus, but may also be derived from other Bacillus sp.Specific examples of contemplated alpha-amylases include the Bacilluslicheniformis alpha-amylase (BLA) shown in SEQ ID NO: 4 in WO 99/19467,the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 5in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase (BSG)shown in SEQ ID NO: 3 in WO 99/19467. In an embodiment of the inventionthe alpha-amylase is an enzyme having a degree of identity of at least60%, preferably at least 70%, more preferred at least 80%, even morepreferred at least 90%, such as at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity to any of the sequences shownas SEQ ID NOS: 1, 2, 3, 4, or 5, respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documentshereby incorporated by reference). Specifically contemplatedalpha-amylase variants are disclosed in U.S. Pat. Nos. 6,093,562,6,297,038 or U.S. Pat. No. 6,187,576 (hereby incorporated by reference)and include Bacillus stearothermophilus alpha-amylase (BSGalpha-amylase) variants having a deletion of one or two amino acid inposition 179 to 182, preferably a double deletion disclosed in WO1996/023873—see e.g., page 20, lines 1-10 (hereby incorporated byreference), preferably corresponding to delta(181-182) compared to thewild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids 179 and 180 usingSEQ ID NO: 3 in WO 99/19467 for numbering (which reference is herebyincorporated by reference). Even more preferred are Bacillusalpha-amylases, especially Bacillus stearothermophilus alpha-amylase,which have a double deletion corresponding to delta(181-182) and furthercomprise a N193F substitution (also denoted I181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO: 3 disclosed in WO 99/19467.

The alpha-amylase may also be a maltogenic alpha-amylase. A “maltogenicalpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is ableto hydrolyze amylose and amylopectin to maltose in thealpha-configuration. A maltogenic alpha-amylase from Bacillusstearothermophilus strain NCIB 11837 is commercially available fromNovozymes A/S, Denmark. The maltogenic alpha-amylase is described inU.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are herebyincorporated by reference.

Bacterial Hybrid Alpha-Amylases

A hybrid alpha-amylase specifically contemplated comprises 445C-terminal amino acid residues of the Bacillus licheniformisalpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676), withone or more, especially all, of the following substitution:

G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the Bacilluslicheniformis numbering). Also preferred are variants having one or moreof the following mutations (or corresponding mutations in other Bacillusalpha-amylase backbones): H154Y, A181T, N190F, A209V and Q264S and/ordeletion of two residues between positions 176 and 179, preferablydeletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO99/19467).

The bacterial alpha-amylase may be added in amounts as are well-known inthe art. When measured in KNU units (described below in the “Materials &Methods”-section) the alpha-amylase activity is preferably present in anamount of 0.5-5,000 NU/9 of DS, in an amount of 1-500 NU/g of DS, ormore preferably in an amount of 5-1,000 NU/g of DS, such as 10-100 NU/gDS.

Fungal Alpha-Amylases

Fungal acid alpha-amylases include acid alpha-amylases derived from astrain of the genus Aspergillus, such as Aspergillus oryzae, Aspergillusniger, or Aspergillus kawachii alpha-amylases.

A preferred acid fungal alpha-amylase is a Fungamyl-like alpha-amylasewhich is preferably derived from a strain of Aspergillus oryzae. In thepresent disclosure, the term “Fungamyl-like alpha-amylase” indicates analpha-amylase which exhibits a high identity, i.e. more than 70%, morethan 75%, more than 80%, more than 85% more than 90%, more than 95%,more than 96%, more than 97%, more than 98%, more than 99% or even 100%identity to the mature part of the amino acid sequence shown in SEQ IDNO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strainAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in theSwiss-prot/TeEMBL database under the primary accession no. P56271 anddescribed in more detail in WO 89/01969 (Example 3). The acidAspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO2004/080923 (Novozymes) which is hereby incorporated by reference. Alsovariants of said acid fungal amylase having at least 70% identity, suchas at least 80% or even at least 90% identity, such as at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 1 in WO 2004/080923 are contemplated. A suitable commerciallyavailable acid fungal alpha-amylase derived from Aspergillus niger isSP288 (available from Novozymes A/S, Denmark).

In a preferred embodiment the alpha-amylase is derived from Aspergilluskawachii and disclosed by Kaneko et al. J. Ferment. Bioeng. 81:292-298(1996) “Molecular-cloning and determination of the nucleotide-sequenceof a gene encoding an acid-stable alpha-amylase from Aspergilluskawachii”; and further as EMBL:#A6008370.

The fungal acid alpha-amylase may also be a wild-type enzyme comprisinga carbohydrate-binding module (CBM) and an alpha-amylase catalyticdomain (i.e., a none-hybrid), or a variant thereof. In an embodiment thewild-type acid alpha-amylase is derived from a strain of Aspergilluskawachii.

Fungal Hybrid Alpha-Amylases

In a preferred embodiment the fungal acid alpha-amylase is a hybridalpha-amylase. Preferred examples of fungal hybrid alpha-amylasesinclude the ones disclosed in WO 2005/003311 or U.S. Patent Publicationno. 2005/0054071 (Novozymes) or U.S. patent application No. 60/638,614(Novozymes) which is hereby incorporated by reference. A hybridalpha-amylase may comprise an alpha-amylase catalytic domain (CD) and acarbohydrate-binding domain/module (CBM) and optional a linker.

Specific examples of contemplated hybrid alpha-amylases include thosedisclosed in U.S. patent application No. 60/638,614 including Fungamylvariant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO:100 in U.S. application No. 60/638,614). Rhizomucor pusillusalpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO: 101 inU.S. application No. 60/638,614) and Meripilus giganteus alpha-amylasewith Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S.application no. 60/638,614).

Other specific examples of contemplated hybrid alpha-amylases includethose disclosed in U.S. Patent Publication no. 2005/0054071, includingthose disclosed in Table 3 on page 15, such as Aspergillus nigeralpha-amylase with Aspergillus kawachii linker and starch bindingdomain.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE from DSM (Gist Brocades), BAN™, TERMAMYL™ SC, FUNGAMYL™,LIQUOZYME™ X and SAN™ SUPER, SANT™ EXTRA L (Novozymes A/S) and CLARASE™L-40,000. DEX-LO™, SPEZYME™ FRED, SPEZYE™ AA, SPEZYME™ Ethyl, andSPEZYME™ DELTA AA (Genencor Int.), and the acid fungal alpha-amylasesold under the trade name SP288 (available from Novozymes NS, Denmark).

An acid alpha-amylases may according to the invention be added in anamount 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS, especially0.3 to 2 AFAU/g DS.

Production of Syrup

The present invention also provides a process of using a glucoamylase ofthe invention for producing syrup, such as glucose and the like, fromstarch-containing material. Suitable starting materials are exemplifiedin the “Starch-containing materials”-section above. Generally, theprocess comprises the steps of partially hydrolyzing starch-containingmaterial (liquefaction) in the presence of alpha-amylase and thenfurther saccharifying the release of glucose from the non-reducing endsof the starch or related oligo- and polysaccharide molecules in thepresence of glucoamylase of the invention.

Liquefaction and saccharification may be carried our as described abovefor fermentation product production.

The glucoamylase of the invention may also be used in immobilized form.This is suitable and often used for producing specialty syrups, such asmaltose syrups, and further for the raffinate stream of oligosaccharidesin connection with the production of fructose syrups, e.g., highfructose syrup (HFS).

Consequently, this aspect of the invention relates to a process ofproducing syrup from starch-containing material, comprising

(a) liquefying starch-containing material in the presence of analpha-amylase,

(b) saccharifying the material obtained in step (a) using a glucoamylaseof the invention.

A syrup may be recovered from the saccharified material obtained in step(b).

Details on suitable conditions can be found above.

Brewing

A glucoamylase of the invention can also be used in a brewing process.The glucoamylases of the invention is added in effective amounts whichcan be easily determined by the skilled person in the art.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and de-scribed herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties. The present invention isfurther described by the following examples which should not beconstrued as limiting the scope of the invention.

Materials & Methods Glucoamylases:

Glucoamylase AN: Glucoamylase derived from Aspergillus niger disclosedin (Boel et al. (1984), EMBO J. 3 (5) p. 1097-1102) and available fromNovozymes NS, Denmark.

Alpha-Amylase:

Alpha-Amylase A: Hybrid alpha-amylase consisting of Rhizomucor pusillusalpha-amylase (SEQ ID NO: 6 herein) with Aspergillus niger glucoamylaselinker (SEQ ID NO: 8 herein) and BD (SEQ ID NO: 10 herein) disclosed asV039 in Table 5 in co-pending International Application no.PCT/US05/46725 (WO 2006/069290).

Yeast RED STAR™ available from Red Star/Lesaffre, USA

Other Materials

pENI2516 is described in WO 2004/069872.

Aspergillus niger MBin118 is disclosed in WO 2004/090155 (see e.g.,Example 11)

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty at Deutshe Sammmlung von Microorganismen andZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE,and given the following accession number:

Deposit Accession Number Date of Deposit Escherichia coli NN49873 DSM18150 3 Apr. 2006

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Media and Reagents:

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

PDA: 39 g/L Potato Dextrose Agar, 20 g/L agar, 50 ml/L glycerol

Methods

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990.

Glucoamylase Activity

Glucoamylase activity may be measured in AGI units or in GlucoamylaseUnits (AG U).

Glucoamylase Activity (AGI)

Glucoamylase (equivalent to amyloglucosidase) converts starch intoglucose. The amount of glucose is determined here by the glucose oxidasemethod for the activity determination. The method described in thesection 76-11 Starch—Glucoamylase Method with Subsequent Measurement ofGlucose with Glucose Oxidase in “Approved methods of the AmericanAssociation of Cereal Chemists”, Vol. 1-2 AACC, from AmericanAssociation of Cereal Chemists, (2000); ISBN: 1-891127-12-8.

One glucoamylase unit (AGI) is the quantity of enzyme which will form 1micro mole of glucose per minute under the standard conditions of themethod.

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch, concentration approx. 16 g dry matter/L.Buffer: Acetate, approx. 0.04 M, pH = 4.3 pH: 4.3 Incubationtemperature: 60° C. Reaction time: 15 minutes Termination of thereaction: NaOH to a concentration of approximately 0.2 g/L (pH ~9)Enzyme concentration: 0.15-0.55 AAU/mL.

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as colorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine.

Glucoamylase Activity (AGU)

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1 M pH:4.30 ± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21 mM Buffer:phosphate 0.12 M; 0.15 M NaCl pH: 7.60 ± 0.05 Incubation temperature:37° C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution, Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ce; and pH 5.6) dextrinizes 5260 mg starch dry substance Merck Amylumsolubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes ALS, Denmark, which folderis hereby included by reference.

Acid Alpha-Amylase Activity

When used according to the present invention the activity of any acidalpha-amylase may be measured in AFAU (Acid Fungal Alpha-amylase Units).Alternatively activity of acid alpha-amylase may be measured in MU (AcidAlpha-amylase Units).

Acid Alpha-Amylase Units (AAU)

The acid alpha-amylase activity can be measured in AAU (AcidAlpha-amylase Units), which is an absolute method. One Acid Amylase Unit(AAU) is the quantity of enzyme converting 1 g of starch (100% of drymatter) per hour under standardized conditions into a product having atransmission at 620 nm after reaction with an iodine solution of knownstrength equal to the one of a color reference.

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch. Concentration approx. 20 g DS/L. Buffer:Citrate, approx. 0.13 M, pH = 4.2 Iodine solution: 40.176 g potassiumiodide + 0.088 g iodine/L City water 15°-20° dH (German degree hardness)pH: 4.2 Incubation temperature: 30° C. Reaction time: 11 minutesWavelength: 620 nm Enzyme concentration: 0.13-0.19 AAU/mL Enzyme workingrange: 0.13-0.19 AAU/mL

The starch should be Lintner starch, which is a thin-boiling starch usedin the laboratory as colorimetric indicator. Lintner starch is obtainedby dilute hydrochloric acid treatment of native starch so that itretains the ability to color blue with iodine. Further details can befound in EP 0140410 B2, which disclosure is hereby included byreference.

Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity may be measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard. 1 AFAU is defined as the amount of enzyme which degrades 5.260mg starch dry matter per hour under the below mentioned standardconditions.

Acid alpha-amylase, an endo-alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase, E.C. 3.2.1.1) hydrolyzesalpha-1,4-glucosidic bonds in the inner regions of the starch moleculeto form dextrins and oligosaccharides with different chain lengths. Theintensity of color formed with iodine is directly proportional to theconcentration of starch. Amylase activity is determined using reversecolorimetry as a reduction in the concentration of starch under thespecified analytical conditions.

Standard Conditions/Reaction Conditions:

Substrate: Soluble starch, approx. 0.17 g/L Buffer: Citrate, approx.0.03 M Iodine (I2): 0.03 g/L CaCl2: 1.85 mM pH: 2.50 ± 0.05 Incubationtemperature: 40° C. Reaction time: 23 seconds Wavelength: 590 nm Enzymeconcentration: 0.025 AFAU/mL Enzyme working range: 0.01-0.04 AFAU/mL

A folder EB-SM-0259.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

EXAMPLES Example 1 DNA Extraction and PCR Amplification of Peniophorarufomarginata Glucoamylase Gene

Aerial hyphae of Peniophora rufomarginata growing on a PDA (PotatoeDextrose Agar) plate were scraped off the plate and used for genomic DNAextraction using FastDNA SPIN Kit for Soil (Qbiogene, USA) according tothe manufacturer's instructions.

PCR reaction was done on genome DNA with the degenerated primers EuAMF1and EuAMR4:

(SEQ ID NO: 11) EuAMF1 5′-ACGTACGGATCCAYTWCTAYWCBTGGACHCGYGA-3′ (SEQ IDNO: 12) EuAMR4 5′-GTACGTAAGCTTRTCYTCRGGGTAVCGDCC-3′

Where D=A or G or T; R=A or G; S=C or G; V=A or C or G; Y=C or T; W=A orT; B=G or C or T; H=A or T or C

The amplification reaction (13 microL) was composed of 1 microL genomeDNA solution, 1 microM primer EuAMF1 (25 pmol/microL), 1 microM primerEuAMR4 (25 pmol/microL), 11 microL Extensor Hi-Fidelity PCR Master Mix(ABgene, UK). The reaction was incubated in a DNA Engine Dyad PTC-0220(MJ Research, USA) programmed as follows: 1 cycle at 94° C. for 5minutes; 20 cycles each at 94° C. for 45 seconds, 65° C. for 45 seconds,with an annealing temperature decline of 1° C. per cycle, and 72° C. for1 minute; followed by 20 cycles at 94° C. for 45 seconds. 48° C. for 45seconds and 72° C. for 1 minute; 1 cycle at 72° C. for 7 minutes; and ahold at 4° C. The PCR product was purified using ExoSAP-IT (USB, USA)according to the manufacturer's instructions and sequenced using theprimers as used in the amplification reaction. The sequence wassubsequently compared to the Aspergillus niger glucoamylase gene,showing that the PCR product encoded a part of a glucoamylase.

Example 2 Cloning of Glucoamylase Gene from Peniophora rufomarginata

From the partial sequence of the Peniophora ruformarginata glucoamylasemore gene sequence was obtained with PCR based gene walking using theVectorette Kit from SIGMA-Genosys. The gene walking was basically doneas described in the manufacturer's protocol. 0.15 micro g genomic DNA ofPeniophora rufomarginata was digested with EcoRI. BamHI, HindIII, andClaI independently. The digested DNA was ligated with the correspondingVectorette units supplied by the manufacture using a DNA Engine DyadPTC-0220 (MJ Research, USA) programmed as follows: 1 cycle at 16° C. for60 minutes: 4 cycles each at 37° C. for 20 minutes, 16° C. for 60minutes. 37° C. for 10 minutes: followed by 1 cycle at 16° C. for 60minutes and a hold at 4° C. The ligation reactions were subsequentdiluted 5 times with sterile water.

PCR reactions with the linker-ligated genome DNA of the Peniophorarufomarginata as template was performed with a DNA Engine Dyad PTC-0220(MJ Research, USA) programmed as follows: 1 cycle at 94° C. for 5minutes; 40 cycles each at 94° C. for 15 seconds, 72° C. for 1 minute,72° C. for 1 minute, 1 cycle at 72° C. for 7 minutes; and a hold at 4°C. using the supplied Vectorette primer and the specific Peniophorarufomarginata AMG primers 50311F1 and 50311R2, respectively, as shownbelow.

50311F1: 5′-CGATTCACACCTGGGACATCAAGG-3′ (SEQ ID NO: 13) 50311R2:5′-AAGACACAGTACCAGACGGGTTGG-3′ (SEQ ID NO: 14)

The amplification reactions (12.5 microL) were composed of 0.5 microL oflinker-ligated genome DNAs, 400 nM Vectorette primer, 400 nM Peniophorarufomarginata specific primer, 11 microL Extensor Hi-Fidelity PCR MasterMix (ABgene, UK).

After the PCR reaction the PCR products were purified using ExoSAP-IT(USB, USA) according to the manufacturers instructions and sequenced andsubsequently compared to the Aspergillus niger glucoamylase gene.

A 1.5 kb amplified band was obtained by the PCR reaction from BamHIdigested genome DNA amplified with the primer 50311R2. Sequencing of thePCR product using this primer showed that it encoded the remaining 350basepairs of the glucoamylase gene in the 5′ direction (N-terminal ofthe encoded protein).

A 1.1 amplified band was obtained by the PCR reaction from ClaI digestedgenome DNA amplified with the primer 50311F1. Sequencing of the PCRproduct using this primer showed that it encoded further 550 basepairsof the glucoamylase gene in the 3′ direction, however not reaching theend of the gene. Therefore, an additional sequencing primer 50311F2,were designed based on the newly obtained additional sequence of theglucoamylase gene. A new DNA-Vectorette ligation and followingamplification set up as described above was set up. A 2 kb PCR productobtained from the HindIII digested genome ligation was sequenced withthe 50311F2 primer, and was shown to encode the remaining part of theglucoamylase gene in the 3′ direction (C-terminal of the encodedprotein).

50311F2 5′ GGTGGCAGCACCGTCGCTGTAACC (SEQ ID NO: 15)

Example 3 Expression of the Glucoamylase Gene from Peniophorarufomarginata

The glucoamylase gene from Peniophora rufomarginata was cloned by PCRusing gDNA as template, Reddy PCR Master Mix (ABgene, UK) and theprimers 50311F3 and 50311R3 as shown below:

50311F3: (SEQ ID NO: 16) 5′ CAGCACGGATCCAAGATGCGTCTCCCACAACTTG 3′50311R3 (SEQ ID NO: 17) 5′ GCATCAAGGCGGCCGCCTAGCGCCAGGAATCGTTGGC 3′

Primer 50311F3 and 50311R3 introduced a BamHI and NotI restrictions sitein the amplified DNA fragment and it was subsequently ligated into theBamHI and NotI restrictions site of the Aspergillus expression vectorpENI2516. The ligation mixture was transformed into E. coli TOP10(Invitrogen, USA) to create the expression plasmid pENI2516AMGNN50311E1.The amplified plasmid was recovered using a QIAprep Spin Miniprep kit(QIAGEN, USA) according to the manufacturer's instructions.

The glucoamylase of pENI2516AMGNN50311E1 was sequenced. Unfortunately, aPCR error occurred in the coding region of the glucoamylase gene. ThePCR error was removed by a second cloning step as described below.

Two PCR reactions were performed. PCR reaction 1 contained 10 ng/microLpENI2516AMGNN50311E1 as template, 0.2 mM dNTP, 1× buffer, 1.5 mM MgCl₂,1 unit DyNAzyme EXT (New England Biolabs, UK), and 1 pmol/microL of eachof the primers NN50311fw1 and NN50311bw2 (see below). The total volumewas 50 microL.

(SEQ ID NO: 18) NN50311fw1: 5′ GCGGATCCACCATGCGTCTCCCACAACTTGGAGTC (SEQID NO: 19) NN50311bw2: 5′ AGCTTGATTACGGGCCAGAGCGTGTTCGTGACPCR reaction 2 contained 10 ng/microL pENI2516AMGNN50311E1 as template.0.2 mM dNTP, 1× buffer, 1.5 mM MgCl₂, 1 unit DyNAzyme EXT (New EnglandBiolabs, UK) and 1 pmol/microL of each of the primers NN50311fw2 andNN50311bw1 (see below). The total volume was 50 microL.

(SEQ ID NO: 20) NN50311fw2: 5′ CGAACACGCTCTGGCCCGTAATCAAGCTTG (SEQ IDNO: 21) NN50311bw1: 5′ GGGCGGCCGCTAGCGCCAGGAATCGTTGGCAGTABoth PCR reaction 1 and PCR reaction 2 were performed with a DNA EngineDyad PTC-0220 (MJ Research, USA) programmed as follows: 1 cycle at 94°C. for 3 minutes; 15 cycles each at 94° C. for 20 seconds, 54° C. for 20seconds and 72° C. for 1 minute. 1 cycle at 72° C. for 5 minutes.

A 0.7 kbp DNA band and a 1.5 kbp DNA band was purified from PCR reaction1 and PCR reaction 2, respectably, using GFX PCR DNA Gel BandPurification Kit (Amersham Biosciences, UK).

A third PCR reaction was done containing 1 micro gram of the purified0.7 kbp DNA band and 1 micro gram of the purified 1.5 kbp DNA band astemplate, 0.2 mM dNTP, 1× buffer, 1.5 mM MgCl₂, 1 unit DyNAzyme EXT (NewEngland Biolabs, UK) and 1 pmol/microL of each of the primers NN50311fw1(SEQ ID NO: 20) and NN50311bw1 (SEQ ID NO: 21). The total volume was 50microL. The PCR reaction was performed with a DNA Engine Dyad PTC-0220(MJ Research, USA) programmed as follows: 1 cycle at 94° C. for 3minutes; 9 cycles each at 94° C. for 20 seconds, 54° C. for 20 secondsand 72° C. for 2 minute, 1 cycle at 72° C. for 5 minutes. The DNA waspurified from the PCR reaction using GFX PCR DNA Gel Band PurificationKit (Amersham Biosciences, UK) and subsequently digested with BamHI andNotI and ligated into the BamHI and NotI restrictions site of theAspergillus expression vector pENI2516. The ligation mixture wastransformed into E. coli TOP10 (Invitrogen, USA) to create theexpression plasmid pENI2516AMGNN50311. The amplified plasmid wasrecovered using JETQUICK Plasmid Miniprep Spin Kit 50 (Genomed, Germany)according to the manufacturer's instructions.

The glucoamylase gene of pENI2516AMGNN50311 was sequenced and verifiedto be identical to the genome sequence.

pENI2516AMGNN50311 was transformed into Aspergillus niger MBin118 andthe glucoamylase expressed using standard method well known in the art.

Example 4 Yeast Propagation

Yeast was propagated prior to fermentation. Corn (yellow dent No. 2) wasground to pass through #45 mesh screen. 200 ml tap water and 1 g ureawere mixed with 300 g corn mash. Penicillin was added to 3 mg/liter. In50 g of the mash slurry, 6.4 microL Glucoamylase AN and 0.024 g dryyeast (from RED START) were added and the pH was adjusted to 5.0. Theyeast slurry was incubated at 32° C. with constant stirring at 300 rpmfor 7 hours in a partially open flask.

One-Step Fermentation Using Peniophora rufomarginata Glucoamylase

All one step ground corn to ethanol treatments were evaluated viamini-scale fermentations. Briefly, 410 g of ground yellow dent corn(with particle size around 0.5 mm) was added to 590 g tap water. Thismixture was supplemented with 3.0 ml 1 g/L penicillin and 1 g of urea.The pH of this slurry was adjusted to 4.5 with 5 N NaOH. DS level wasdetermined to be around 35 wt, % (The actual DS was measured with anIR-200 moisture analyzer from Denver Instrument Co.). Approximately 5 gof this slurry was added to 20 ml vials. Each vial was dosed with theappropriate amount of enzyme followed by addition of 200 micro litersyeast propagate per 5 g slurry. Actual enzyme dosages were based on theexact weight of corn slurry in each vial, Vials were closed andincubated at 32° C. immediately. 9 replicate fermentations of eachtreatment were run. Three replicates were selected for 24 hours, 48hours and 70 hours time point analysis. Vials were vortexed at 24, 48and 70 hours and analyzed by HPLC. The HPLC preparation consisted ofstopping the reaction by addition of 50 microliters of 40% H₂SO₄,centrifuging, and filtering through a 0.45 micrometer filter. Sampleswere stored at 4° C. prior to analysis.

Agilent™ 1100 HPLC system coupled with RI detector was used to determineethanol and sugars. The HPLC system consists of a degasser, quat-pump,cooled autosampler and heated column compartment. The separation columnwas aminex HPX-87H ion exclusion column (300 mm×7.8 mm) from BioRad™,which links to 30 mm×4.6 mm micro-guard cation-H cartridge guard column.10 microL sample was injected at the flow rate of 0.6 ml/min. The mobilephase was 5 mM H₂SO₄. The column was kept at 65° C. and RI detector at50° C. The total run time was 25 min per sample.

The results are shown in the table below. Increase of P. ruformarginataglucoamylase results in increase of ethanol yield. High ethanol yield isachieved when P. ruformarginata glucoamylase is used together withalpha-amylase Alpha-Amylase A in one step corn to ethanol process.

Ethanol (% w/v) P. rufomarginata AMG Alpha-Amylase A 24 48 70 (mgenzyme/g DS) (mg enzyme/g DS) hours hours hours 1 0 — 1.75 2.07 2.34 20.02 — 2.47 3.52 4.55 3 0.04 — 2.98 4.25 5.62 4 0.08 3.79 5.75 7.28 5 00.04 6.78 10.90 13.25 6 0.02 0.04 7.47 11.72 13.57 7 0.04 0.04 7.7712.49 14.66 8 0.08 0.04 8.46 13.14 15.50

1. A polypeptide having glucoamylase activity, selected from the groupconsisting of: (a) a polypeptide having an amino acid sequence which hasat least 60% identity with amino acids for mature polypeptide aminoacids 1 to 558 of SEQ ID NO: 2; (b) a polypeptide which is encoded by anucleotide sequence (i) which hybridizes under at least low stringencyconditions with nucleotides 61 to 2301 of SEQ ID NO: 1, or (ii) whichhybridizes under at least low stringency conditions with the cDNAsequence contained in nucleotides 61 to 1734 of SEQ ID NO: 3, or (iii) acomplementary strand of (i) or (ii); (c) a variant comprising aconservative substitution, deletion, and/or insertion of one or moreamino acids of amino acids 1 to 558 of SEQ ID NO:
 2. 2. A polypeptidehaving carbohydrate-binding affinity, selected from the group consistingof: (a) i) a polypeptide comprising an amino acid sequence which has atleast 60% identity with amino acids 464 to 558 of SEQ ID NO: 2; (b) apolypeptide which is encoded by a nucleotide sequence which hybridizesunder low stringency conditions with a polynucleotide probe selectedfrom the group of (i) the complementary strand of nucleotides 1845-2301of SEQ ID NO: 1; (ii) the complementary strand of nucleotides 1450-1734of SEQ ID NO: 3; (c) a fragment of (a) or (b) that has carbohydratebinding affinity.
 3. A polynucleotide having a nucleotide sequence whichencodes for the polypeptide of claim
 1. 4. A nucleic acid constructcomprising the polynucleotide of claim 3 operably linked to one or morecontrol sequences that direct the production of the polypeptide in anexpression host.
 5. A recombinant expression vector comprising thenucleic acid construct of claim
 4. 6. A recombinant host cell comprisingthe nucleic acid construct of claim
 4. 7. (canceled)
 8. A method forproducing a polypeptide of claim 1 comprising (a) cultivating a hostcell comprising a nucleic acid construct comprising a nucleotidesequence encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. 9.(canceled)
 10. A process for producing a fermentation product fromstarch-containing material comprising the steps of: (a) liquefyingstarch-containing material; (b) saccharifying the liquefied materialusing a glucoamylase of claim 1; (c) fermenting the saccharifiedmaterial using a fermenting organism.
 11. A process for producing afermentation product from starch-containing material comprising: (a)saccharifying starch-containing material with a glucoamylase of claim 1,at a temperature below the initial gelatinization temperature of saidstarch-containing material, (b) fermenting using a fermenting organism.12-16. (canceled)
 17. A process of producing syrup fromstarch-containing material, comprising (a) liquefying starch-containingmaterial, preferably in the presence of an alpha-amylase, (b)saccharifying the material obtained in step (a) using a glucoamylase ofclaim.
 18. The process of claim 17, further comprising refining,conversion and/or recovery of the syrup. 19-21. (canceled)
 22. Acomposition comprising a glucoamylase of claim
 1. 23. The composition ofclaim 22, further comprising an alpha-amylase. 24-29. (canceled)